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The Journal of Cell Biology, Vol 112, 1133-1141, Copyright © 1991 by The Rockefeller University Press

ARTICLES

Isolation of functional, coated, endocytic vesicles

PG Woodman and G Warren
Cell Biology Laboratory, Imperial Cancer Research Fund, London, England.

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N- ethyl maleimide.
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